Project Summary Antibiotic courses of two or more weeks cause neutropenia and other cytopenias in up to 15% of patients, which can lead to increased medical costs and mortality. Given that 269 million prescriptions of antibiotics were prescribed to patients in the United States in 2015, these adverse hematological events and their downstream consequences represent a major clinical problem. Thus, the overarching goal of this study is to understand the mechanisms by which the microbiota promote normal hematopoiesis. We and others have shown that the microbiome and its byproducts are necessary for maintaining normal hematopoiesis and that depletion of the microbiota in animal models, such as after antibiotic administration, causes cytopenias. Our lab has established a mouse model of antibiotic-induced bone marrow suppression that demonstrated that bacterial microbiome-mediated hematopoiesis is Stat1-dependent. Our new preliminary data indicate that the microbiome promotes Stat1 signaling via type I interferons (IFN). However, the specific molecular mechanisms by which the microbiome contributes to steady-state hematopoiesis remain poorly understood. A recent study demonstrated that hematopoiesis in germ-free mice, which display hematopoietic abnormalities similar to antibiotic-treated mice, can be rescued by oral administration of NOD1 ligand (NOD1L). Our preliminary data suggest that bacterially-derived molecules such as NOD1L and desaminotyrosine (DAT), a microbial metabolite, may be recognized by the bone marrow to maintain normal blood production. This proposal will test the hypothesis that microbial byproducts utilize immune-related signaling pathways such as Stat1 and Nod1 to regulate steady-state hematopoiesis. In order to test this hypothesis, we first will validate NOD1L and DAT as novel therapeutic agents for antibiotic-mediated neutropenia. Next, we will compare differences in the metabolomic profiles of neutropenic antibiotic-treated wild-type mice and their non-neutropenic counterparts using liquid chromatography and mass spectrometry to identify other microbial cues that maintain normal hematopoiesis. While these studies will identify the bacterial byproducts that support steady-state hematopoiesis, which cell types are affected by these signals remains unknown. For instance, NOD1L is known to induce cytokine production in mesenchymal stromal cells, an important component of the bone marrow microenvironment, while IFNs can induce differentiation in HSCs. Therefore, we also will identify the cell types influenced by administration of NOD1L and DAT, as well as the mechanisms controlling granulopoiesis. Finally, we will define the link between the microbiome and hematopoiesis in patients by analyzing the bacterial and metabolomic compositions of antibiotic-treated patients with and without neutropenia. These studies will elucidate the mechanisms by which the microbiome regulates hematopoiesis and may identify novel therapeutics to rescue neutropenia induced by long-term antibiotic treatment.